Telomeres at chromosome ends shorten during cell division and aging in humans. Critically short dysfunctional telomeres trigger cell senescence or apoptosis, which contributes to aging-related diseases and degeneration. Oxidative stress accelerates telomere shortening, and generates reactive oxygen species (ROS), which are particularly damaging to telomeric TTAGGG repeat sequences. The goals of this proposal are to 1) measure the response of base excision repair (BER) proteins to DNA damage at telomeric regions compared with non- telomere regions; and 2) to define the impact of oxidized telomeric DNA on telomere integrity and telomere functions in preventing cell senescence and cell death. A critical barrier to investigating DNA damage and repair at telomeres has been an inability to target damage to the telomeres. To overcome this obstacle we developed a highly innovative system for confining ROS-induced DNA damage to defined regions in the genome. For this we fused KillerRed (KR) protein, a photosensitizer that generates ROS upon light irradiation, to a Tet-repressor (tetR) protein that binds to a single engineered site within condensed chromatin. We established that oxidative damage occurred only at the tetR bound site. To target telomeres we fused KR to the telomere binding protein TRF1 and showed that this restricts ROS-induced damage to the telomeres. In Aim 1, we will combine the KR system with confocal microscopy to visualize the real time damage response of BER proteins to targeted oxidative damage in telomeric and non-telomeric genomic regions, with 3D resolution in a single cell nucleus. We will measure the mobilization kinetics of various GFP-tagged BER proteins, and the protein domains required for the response, to ROS damage at the TRF1 bound telomeric sites compared with the tetR bound non-telomeric site. In Aim 2, we will test several endpoints of telomere damage and dysfunction, including cellular senescence and apoptosis, after ROS production by activating KR with light exposure in cells that stably express KR-TRF1, compared with cells expressing HcRed-TRF1 as a control. This study will include both bulk cell population and single cell experiments. Using the microscopy system to target KR activation to defined numbers of KR-TRF1 foci, we will measure the average number of oxidized telomeres required to trigger apoptosis or senescence. Successful completion of the project will provide crucial insights into how oxidative stress accelerates telomere shortening, and how telomeric oxidative base damage impacts telomere function and promotes cell senescence. The results will pave the way for future work examining how telomeric damage and repair change with age and vary with cell type, and should lead to new strategies for preserving telomere function to promote healthy aging. !